Attenuated T.sub.2 relaxation by mutual cancellation of dipole--dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. In this patent specification, the numbers in parenthesis refer to the references listed following the description of the preferred embodiment. The following definitions are used throughout this specification for reasons of conciseness:
Abbreviations: NMR, nuclear magnetic resonance; rf, radio-frequency; 2D, two-dimensional; FID, free induction decay; DD, dipole--dipole; CSA, chemical shift anisotropy; COSY, correlation spectroscopy; TROSY, transverse relaxation-optimized spectroscopy; PFG, pulsed field gradient; ftz homeodomain; fushi tarazu homeodomain polypeptide of 70 amino acid residues, with the homeodomain in positions 3-62.
Fast transverse relaxation of .sup.1 H, .sup.15 N and .sup.13 C by dipole--dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. TROSY (Transverse Relaxation-Optimized SpectroscopY) is a new approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example. a TROSY-type two-dimensional .sup.1 H, .sup.15 N-correlation experiment with a uniformly .sup.15 N-labeled protein in a DNA complex of molecular weight 17 kDa at a .sup.1 H frequency of 750 MHz showed that .sup.15 N relaxation during .sup.15 N chemical shift evolution and .sup.1 H.sup.N relaxation during signal acquisition are both significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional .sup.1 H, .sup.15 N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for .sup.15 N and 15 Hz for .sup.1 H.sup.N at 4.degree. C. Since the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a .sup.15 N-labeled protein of 150 kDa at 750 MHz and 20.degree. C. one predicts residual linewidths of 10 Hz for .sup.15 N and 45 Hz for .sup.1 H.sup.N, and for the corresponding uniformly .sup.15 N, .sup.2 H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.
Nuclear magnetic resonance (NMR) spectroscopy with proteins based on observation of a small number of spins with outstanding spectral properties, which may either be present naturally or introduced by techniques such as site-specific isotope labeling, yielded biologically relevant information on human hemoglobin (M=65000) as early as 1969 (Shulman, R. G., Ogawa, S., Wurthrich, K., Yamane, T., Peisach, J. & Blumberg, W. E. (1996) Science 165, 251-257), and subsequently also for significantly larger systems such as, for example, immunoglobulins (Arata, Y., Kato, K., Takahashi, H. & Shimada, I. (1994) Methods in Enzymology 239 440-464). In contrast, the use of NMR for de novo structure determination (Wuthrich, K. (1996) NMR of Proteins and Nucleic Acids (Wiley, New York); Wuthrich, K. (1995) NMR in Structure Biology (World Scientific, Singapore)) has so far been limited to relatively small molecular sizes, with the largest NMR structure below molecular weight 30000. Although NMR in structural biology may, for practical reasons of coordinated use with X-ray crystallography (Wuthrich, K. (1995) Acta Cryst. D 51, 249-270), focus on smaller molecular sizes also in the future, considerable effort goes into attempts to extend the size limit to bigger molecules (for example, Shan, X., Gardner, K. H., Munhandiram, D. R., Rao, N. S., Arrowsmith, C. H. & Kay, L. E. (1996) J. Am. Chem. Soc. 118, 6570-6579; Wagner, G. (1993) J. Biomol, NMR 3, 375-385; Nietlispach, D., Clowes, R. T., Broadhurst, R. W., Ito, Y., Keeler, J., Kelly, M., Ashurst, J., Oschkinat, H., Domaille, P. J. & Laue, E. D. (1996) J. Am. Chem. Soc. 118, 407-415). Here we introduce "transverse relaxation-optimized spectroscopy" (TROSY) and present experimental data and theoretical considerations showing that this novel approach is capable of significantly reducing transverse relaxation rates and thus overcomes a key obstacle opposing solution NMR of larger molecules (Wagner, G. (1993) J. Biomol. NMR 3, 375-385).
At the high magnetic fields typically used for studies of proteins and nucleic acids, chemical shift anisotropy interaction (CSA) of .sup.1 H, .sup.15 N and .sup.13 C nuclei forms a significant source of relaxation in proteins and nucleic acids, in addition to dipole--dipole (DD) relaxation. This leads to increase of the overall transverse relaxation rates with increasing polarizing magnetic field, B.sub.0. Nonetheless, transverse relaxation of amide protons in larger proteins at high fields has been successfully reduced by complete or partial replacement of the non-labile hydrogen atoms with deuterons and, for example, more than 90% of the .sup.15 N, .sup.13 C.sup..alpha. and .sup.1 H.sup.N chemical shifts were thus assigned in the polypeptide chains of a protein-DNA complex of size 64000 (Shan, X., Gardner, K. H., Munhandiram, D. R., Rao, N. S., Arrowsmith, C. H. & Kay, L. E. (1996) J. Am. Chem. Soc. 118, 6570-6579). TROSY uses spectroscopic means to further reduce T.sub.2 relaxation based on the fact that cross-correlated relaxation caused by DD and CSA interference gives rise to different relaxation rates of the individual multiplet components in a system of two coupled spins 1/2, I and S, such as, for example, the .sup.15 N--.sup.1 H fragment of a peptide bond (Gueron, M., Leroy, J. L. & Griffey, R. H. (1983) J. Am. Chem. Soc. 105, 7262-7266; Tjandra, N., Szabo, A. & Bax, A. (1996) J. Am. Chem. Soc. 118, 6986-6991). Theory shows that at .sup.1 H frequencies near 1 GHz nearly complete cancellation of all transverse relaxation effects within a .sup.15 N--.sup.1 H moiety can be achieved for one of the four multiplet components. TROSY observes exclusively this narrow component, for which the residual linewidth is then almost entirely due to DD interactions with remote hydrogen atoms in the protein. These can be efficiently suppressed by .sup.2 H-labeling, so that in TROSY-type experiments the accessible molecular size for solution NMR studies is no longer primarily limited by T.sub.2 relaxation.